The long term goal of this project is to study, isolate, and characterize monocyte cytotoxicity factor (MCF) produced by human T lymphocytes which induces cytotoxicity against human tumors. This factor will be useful in probing the immunobiology of T-lymphocyte/monocyte interactions, and will be of value clinically in augmenting host defense against tumors. OKT4+ lymphocytes from patients with Sezary's syndrome, a T-lymphocyte proliferative disorder known to display helper function were immortalized by fusion with the continuous T-cell line CEM.8aza(r).C. Two clones which produced MCF were uniformly OKT4+, and OKT8-, unlike the parent CEM.8aza(r).C. The biologic activity was not neutralized by antisera to native IFN-Gamma or a 20-peptide N-terminal fragment of IFN-Gamma. Moreover, supernatants concentrated by chromatography on Matrix Gel Red A, which binds our MCF, had no detectable IFN activity. Activity was retained over a wide pH range (2-7.4,4hrs), unlike IFN-Gamma. Our factor was stable at 4, 37, and 60C(2hrs), but was inactivated at 100C, and was inactivated by chymotrypsin, but not trypsin. We plan the following experiments: 1) Conditions for low serum or serum-free culture of our clones will be developed to simplify purification. 2) Sensitivity to proteases, RN- and DNases, and solubility in organic solvents will be determined. 3) Purification will proceed along two pathways simultaneously: a) concentration by either (NH4)2SO4, lyophilization, or dye ligand chromatography. b) followed by fractionation by size exclusion, hydrophobic chromatography, and 2-D PAGE. 4) Production of a murine monoclonal antibody which will then be coupled to Sepharose for used in affinity chromatography. Once prepared studies of MCF could be extended to study binding to various classes of mononuclear phagocytes, and to isolate putative receptor structures. Antibody could be used to develop a radioimmunoassay for MCF to measure activity in preparations and in patients' sera. Moreover, the role of MCF in preventing experimental tumor metastases could be studied.